The present invention relates to the field of plant biotechnology. More specifically, it concerns methods for incorporating genetic components into a plant via genetic engineering techniques. In particular, provided herein are systems for genetically transforming corn.
During the past decade, it has become possible to transfer genes from a wide range of organisms to crop plants by recombinant DNA technology. This advance has provided enormous opportunities to improve plant resistance to pests, disease and herbicides, and to modify biosynthetic processes to change the quality of plant products for food, feed and industrial uses.
Agrobacterium-mediated transformation is one method for introducing a desired genetic element into a plant and is achieved through the use of a genetically engineered soil bacterium belonging to the genus Agrobacterium. Several-Agrobacterium species mediate the transfer of a specific DNA known as “T-DNA” that can be genetically engineered to carry a desired piece of DNA into many plant species. The major events marking the process of T-DNA mediated pathogenesis are induction of virulence genes, processing and transfer of T-DNA.
Agrobacterium-mediated genetic transformation of plants involves several steps. The first step, in which the virulent Agrobacterium and plant cells are first brought into contact with each other, is generally called “inoculation”. Following the inoculation step, the Agrobacterium and plant cells/tissues are usually grown together for a period of several hours to several days or more under conditions suitable for growth and T-DNA transfer. This step is termed “co-culture”. Following co-culture and T-DNA delivery, the plant cells are often treated with bactericidal or bacteriostatic agents to kill the Agrobacterium. If this is done in the absence of any selective agents to promote preferential growth of transgenic versus non-transgenic plant cells, then this is typically referred to as the “delay” step. If done in the presence of selective pressure favoring transgenic plant cells, then it is referred to as a “selection” step. When a “delay” is used, it is typically followed by one or more “selection” steps. Both the “delay” and “selection” steps typically include bactericidal or bacteriostatic agents to kill any remaining Agrobacterium cells because the growth of Agrobacterium cells is undesirable after the infection (inoculation and co-culture) process.
Another widely used technique to genetically transform plants involves the use of microprojectile bombardment. In this process, a nucleic acid containing the desired genetic elements to be introduced into the plant is deposited on small metallic, e.g., gold or tungsten, particles, which are then delivered at a high velocity into the plant tissue or plant cells. Cells containing the desired genetic elements are then placed in tissue culture and transformed cells selected through the use of one or more selection system that has been incorporated into the genetic elements transformed into the plant.
The major deficiencies in current plant transformation systems include but are not limited to the production efficiency of the system, and transformation variability due to genotype or species diversity and explant limitations. In particular, there is a continuing need in the field of plant biotechnology to provide more efficient transformation methods suitable for high capacity production of economically important plants, particularly elite cultivars.